This invention relates generally to a method of monoclonal antibody production and specifically to the simultaneous in vitro affinity optimization of multiple distinct domains of a variable region of a monoclonal antibody.
The War on Cancer is entering its third decade and recent years have shown tremendous progress in the understanding of cancer development and progression yet there has been only marginal decreases in death rates from most types of cancer. Standard chemotherapy and radiation therapy generally involve treatment with therapeutic agents that impact not only cancer cells but other highly proliferative cells of the body, often leading to debilitating side effects. Thus, it is desirable to identify therapeutic agents with a higher degree of specificity for the carcinogenic lesion.
The discovery of monoclonal antibodies (mabs) in the 1970's provided great hope for the reality of creating therapeutic molecules with high specificity. Antibodies that bind to tumor antigens would provide specific targeting agents for cancer therapy. However, while the development of monoclonal antibodies has provided a valuable diagnostic reagent, certain limitations restrict their use as therapeutic entities.
A limitation encountered when attempts are made to use mAbs as therapeutic agents is that since mAbs are developed in non-human species, usually mouse, they elicit an immune response in human patients. Chimeric antibodies join the variable region of the non-human species, which confers binding activity, to a human constant region. However, the chimeric antibody is often still immunogenic and it is therefore necessary to further modify the variable region.
One modification is the grafting of complementarity-determining regions, (CDRs) which are in part antigen binding onto a human antibody variable framework. However, this approach is imperfect because CDR grafting often diminishes the binding activity of the resulting humanized mAb. Attempts to regain binding activity require laborious, step-wise procedures which have been pursued essentially by a trial and error type of approach. For example, one difficulty in regaining binding affinity is because it is difficult to predict which framework residues serve a critical role in maintaining antigen binding affinity and specificity. Consequently, while antibody humanization methods that rely on structural and homology data are used, the complexity that arises from the large number of framework residues potentially involved in binding activity has prevented success.
Combinatorial methods have been applied to restore binding affinity, however, these methods require sequential rounds of mutagenesis and affinity selection that can both be laborious and unpredictable.
Thus, there exists a need for efficient and reliable methods for producing human monoclonal antibodies which exhibit comparable or enhanced binding affinities to their non-human counterparts. The present invention satisfies this need and provides related advantages as well.